Of fundamental importance to bridging the gap between biochemistry and cell biology is the ability to study proteins in a living cell. A great deal of progress has been made in this regard both on the chemistry side and the imaging side. Many new chemical tools are now in place to visualize proteins in vivo as well as single molecule and ensemble visualization techniques to utilize these tools. However, to date no general and broadly applicable tool exists to label individual proteins in a specific and non-perturbing manner. Moreover, the existing labels do not possess the required photophysical properties for detailed probing of protein dynamics, localization, and interaction with other members of the cellular environment. Current shortfalls of existing techniques include large fluorophores, toxic reagents, cell impermeability, slow maturation times, or poor photophysical properties. A general protein labeling tool that addresses each of these problems and allows incorporation of multiple labels for fluorescence resonance energy transfer or spin-spin coupling studies would provide an invaluable tool to bridge the gap between biochemistry, genetics, and cell biology. One common protein labeling strategy uses an organism's biosynthetic machinery to insert the desired probe. We have previously demonstrated the use of fatty acid biosynthetic machinery in vivo to incorporate non-canonical phosphopantetheine analogues onto truncated modular carrier proteins. This methodology has provided a general labeling scheme, but to date the inefficiency of this system, caused by competition from the natural ligand, coenzyme A (CoA), has prohibited general application. In order to create a robust tool, we will investigate a series of unnatural pantothenate analogues that are not recognized by the first and last enzymes of this biosynthetic pathway. We will follow with directed evolution of these two enzymes to accept a set of non-canonical substrates in order to provide a protein labeling paradigm that will be fully orthogonal to endogenous machinery. These tools will allow specific and efficient incorporation of desired labels without competing side reactions. After implementation in a mammalian cell culture, we will probe the role of I:B to strip NF:B from DNA in the nucleus of living cells through ensemble and single-molecule FRET techniques. Public Health Relevance: The proposed research will add a general tool to probe protein function in vivo. All organisms require properly functioning and correctly localized gene products to survive, and following the production, movement, and interactivity of gene products remains an important goal for the study of human diseases caused by improper protein import and export, incorrect folding to a functional tertiary structure, and formation of deleterious higher order aggregates or structures. Here we propose the study of a novel protein labeling system that harnesses an existing metabolic pathway to attach a chemical probe onto proteins within living cells. Selective application of these probes will allow us to study important protein activity in living cells, and here we propose to use these tools in the study of gene activation by regulatory proteins.